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• W2022025232 abstract "GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (·NO) or hydrogen peroxide (H2O2), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO–) and hypochlorous acid (HOCl). For the exposure of 17.5 μm GroEL to 100–250 μm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met111 and Met114 to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO– suggests that this protein may be a target for bacterial killing by phagocytes. GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (·NO) or hydrogen peroxide (H2O2), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO–) and hypochlorous acid (HOCl). For the exposure of 17.5 μm GroEL to 100–250 μm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met111 and Met114 to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO– suggests that this protein may be a target for bacterial killing by phagocytes. GroEL and its eukaryotic analog, heat shock protein 60 (Hsp60), 1The abbreviations used are: Hsp60, heat shock protein 60; CID, collision-induced dissociation; Cpn60, chaperonin 60; DEA/·NO, diethylamine/nitric oxide; DTT, dithiothreitol; H2O2, hydrogen peroxide; HOCl, hypochlorous acid; HPLC, high performance liquid chromatography; MDH, malate dehydrogenase; MS, mass spectrometry; MsrB/A, methionine sulfoxide reductase B/A; mt-Cpn60, mitochondrial chaperonin 60; ·NO, nitric oxide;O2⋅¯, superoxide; ONOO–, peroxynitrite. 1The abbreviations used are: Hsp60, heat shock protein 60; CID, collision-induced dissociation; Cpn60, chaperonin 60; DEA/·NO, diethylamine/nitric oxide; DTT, dithiothreitol; H2O2, hydrogen peroxide; HOCl, hypochlorous acid; HPLC, high performance liquid chromatography; MDH, malate dehydrogenase; MS, mass spectrometry; MsrB/A, methionine sulfoxide reductase B/A; mt-Cpn60, mitochondrial chaperonin 60; ·NO, nitric oxide;O2⋅¯, superoxide; ONOO–, peroxynitrite. are highly sequence-related members of the Group I subclass of chaperonin 60 (Cpn60) (1Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2775) Google Scholar). These proteins assist the folding of newly synthesized polypeptides (GroEL) or translocated preproteins (mitochondrial Hsp60). The functional unit of GroEL (and of most Cpn60 proteins) is a sandwich of two heptameric rings, which are stacked end to end. Depending on the protein substrate, different ligands such as K+, Mg2+, ATP, and the cofactor GroES (or Hsp10) may be required for proper folding. Following the trapping of an unfolded or misfolded protein substrate in the hydrophobic interior of GroEL, the binding of ATP and GroES causes a conformational transition, which changes the interior surface properties from hydrophobic to hydrophilic, thus triggering protein folding (1Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2775) Google Scholar, 2Netzer W.J. Hartl F.U. Trends Biochem. Sci. 1998; 23: 68-73Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Mammalian Hsp60 differs from GroEL in that it forms stable and functional heptameric rings in the absence of ATP and its cofactor Hsp10 (3Levy-Rimler G. Viitanen P. Weiss C. Sharkia T. Greenberg A. Niv A. Lustig A. Delarea Y. Azem A. Eur. J. Biochem. 2001; 268: 3465-3472Crossref PubMed Scopus (85) Google Scholar). Our rationale for investigating the oxidative inactivation of GroEL is 2-fold: (i) its potential involvement in bacterial killing by phagocytes and (ii) a potential role for its analog, Hsp60, in an inflammatory and proapoptotic response during cardiovascular disease, as described below. Hsp60 proteins play an important role in the cellular protection against oxidative stress (4Cabiscol E. Piulats E. Echaves P. Herrero E. Ros J. J. Biol. Chem. 2000; 275: 27393-27398Abstract Full Text Full Text PDF PubMed Google Scholar, 5Cabiscol E. Bellí G. Tamarit J. Echaves P. Herrero E. Ros J. J. Biol. Chem. 2002; 277: 44531-44538Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Studies with mutant strains of Saccharomyces cerevisiae exposed to various oxidants show that a decrease in Hsp60 expression results in an increased sensitivity toward oxidative stress (reduced cell viability) and increased levels of oxidized mitochondrial proteins, including Hsp60 itself (5Cabiscol E. Bellí G. Tamarit J. Echaves P. Herrero E. Ros J. J. Biol. Chem. 2002; 277: 44531-44538Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). An important question, which has not been addressed, is whether the oxidation of Hsp60 leads to its inactivation. If so, Hsp60 oxidation would further decrease the levels of active Hsp60 eventually leading to a more pronounced sensitivity of cells toward oxidative stress. Here, we will provide evidence that the bacterial analog of Hsp60, GroEL, is fairly insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (·NO) or hydrogen peroxide (H2O2). In contrast, GroEL is efficiently modified and inactivated through reactive species generated by phagocytes, such as peroxynitrite (ONOO–) and hypochlorous acid (HOCl). Such sensitivity toward phagocyte-derived oxidants suggests that GroEL oxidation may represent an effective mechanism for bacterial killing by neutrophils and macrophages. Peroxynitrite, the product of ·NO and superoxide (O2.), is highly toxic toward bacteria (6Zhu L. Gunn C. Beckman J.S. Arch. Biochim. Biophys. 1992; 298: 452-457Crossref PubMed Scopus (415) Google Scholar, 7Brunelli L. Crow J.P. Beckman J.S. Arch. Biochim. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (263) Google Scholar). Hypochlorous acid is generated from chloride ions (Cl–) and H2O2 by the heme enzyme myeloperoxidase (8Thomas E.L. Infect. Immun. 1979; 23: 522-531Crossref PubMed Google Scholar, 9Sips H.J. Hamers M.N. Infect. Immun. 1981; 31: 11-16Crossref PubMed Google Scholar, 10Podrez E.A. Abu-Soud H.M. Hazen S.L. Free Radic. Biol. Med. 2000; 28: 1717-1725Crossref PubMed Scopus (529) Google Scholar). A prominent role for myeloperoxidase in bacterial killing has been proposed (11Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar, 12Klebanoff S.J. Shepard C.C. Infect. Immun. 1984; 44: 534-536Crossref PubMed Google Scholar, 13Rosen H. Klebanoff S.J. Infect. Immun. 1985; 47: 613-618Crossref PubMed Google Scholar, 14Klebanoff S.J. Waltersdorph A.M. Rosen H. Methods Enzymol. 1984; 105: 399-403Crossref PubMed Scopus (208) Google Scholar, 15Rosen H. Rakita R.M. Waltersdorph A.M. Klebanoff S.J. J. Biol. Chem. 1987; 242: 15004-15010Abstract Full Text PDF Google Scholar, 16Albrich J.M. McCarthy C.A. Hurst J.K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 210-214Crossref PubMed Scopus (384) Google Scholar) and confirmed in vivo involving a mouse model of polymicrobial sepsis (17Gaut J.P. Yeh G.C. Tran H.D. Byun J. Henderson J.P. Richter G.M. Brennan M.L. Lusis A.J. Belaaouaj A. Hotchkiss R.S. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11961-11966Crossref PubMed Scopus (221) Google Scholar). Aberrant Hsp60 may also play a role during inflammation of human tissue. Inflammatory processes contribute to the pathogenesis of some cardiovascular diseases such as atherosclerosis (18Berliner J.A. Heinecke J.W. Free Radic. Biol. Med. 1996; 20: 707-727Crossref PubMed Scopus (1272) Google Scholar, 19Wick G. Xu Q. Exp. Gerontol. 1999; 34: 559-566Crossref PubMed Scopus (53) Google Scholar, 20Napoli C. Nigris F.D. Palinski W. J. Cell. Biochem. 2001; 82: 674-682Crossref PubMed Scopus (220) Google Scholar, 21Brennan M.-L. Hazen S.L. Amino Acids. 2003; 25: 365-374Crossref PubMed Scopus (47) Google Scholar). Mechanical shear stress, such as observed in atherosclerotic aorta, triggers the expression of Hsp60, which stimulates the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and interleukin-6 (22Kol A. Bourcier T. Lichtman A.H. Libby P. J. Clin. Invest. 1999; 103: 571-577Crossref PubMed Scopus (464) Google Scholar), thus promoting an inflammatory response (19Wick G. Xu Q. Exp. Gerontol. 1999; 34: 559-566Crossref PubMed Scopus (53) Google Scholar). Interestingly, stressed aortic endothelial cells present Hsp60 on the cell surface (23Xu Q. Schett G. Seitz C.S. Hu Y. Gupta R.S. Wick G. Circ. Res. 1994; 75: 1078-1085Crossref PubMed Scopus (153) Google Scholar). A similar observation was made for oxidatively stressed myocytes (hypoxia/reoxygenation), where translocation of cytosolic Hsp60 to the cell surface induced apoptosis (24Gupta S. Knowlton A.A. Circulation. 2002; 106: 2727-2733Crossref PubMed Scopus (139) Google Scholar). In the cytosol, Hsp60 complexes the propaptotic protein bax, and upon translocation of Hsp60 to the cell surface, the Hsp60/bax complex dissociates and bax relocates into the mitochondria (24Gupta S. Knowlton A.A. Circulation. 2002; 106: 2727-2733Crossref PubMed Scopus (139) Google Scholar). The molecular reason for Hsp60 translocation is not known. Coronary artery disease correlates with increased levels of myeloperoxidase (25Zhang R. Brennan M.L. Fu X. Aviles R.J. Pearce G.L. Penn M.S. Topol E.J. Sprecher D.L. Hazen S.L. J. Am. Med. Assoc. 2001; 286: 2136-2142Crossref PubMed Scopus (763) Google Scholar) and myeloperoxidase-dependent markers for protein oxidation have been detected in low density lipoprotein isolated from human atherosclerotic intima (26Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (752) Google Scholar). Based on the sensitivity of GroEL to HOCl demonstrated in this paper, it is possible that myeloperoxidase-derived reactive species target human Hsp60 in the aorta and that oxidative modification and inactivation of Hsp60 contribute to the pathogenesis of atherosclerosis. The GroE molecular chaperonins of Escherichia coli were isolated from the lysate of cells containing the appropriate overexpression plasmids (gifts from Dr. Edward Eisenstein (27Lin Z. Schwarz F.P. Eisenstein E. J. Biol. Chem. 1995; 270: 1011-1014Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and Dr. Lorimer (28Goloubinoff P. Gatenby A. Lorimer G.H. Nature. 1989; 337: 44-47Crossref PubMed Scopus (524) Google Scholar), respectively), as described by Voziyan and Fisher (29Voziyan P.A. Fisher M.T. Protein Sci. 2000; 9: 2405-2412Crossref PubMed Scopus (43) Google Scholar). Because GroEL and GroES do not contain tryptophan residues, the removal of tryptophan containing contaminants, as assayed by second derivative analysis of the absorption spectra and tryptophan fluorescence, was used as a criterion for purity of the chaperonin preparations in addition to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Stock solutions (∼10 mg/ml) were kept at 4 °C in 60% ammonium sulfate. Before each set of experiments, GroEL was exchanged into buffer A, consisting of 50 mm Tris, pH 7.5, 10 mm magnesium chloride, and 50 mm potassium chloride using Pierce Slide-A-Lyzer 3K Dialysis Cassettes. This was followed by exchange into 50 mm sodium phosphate, pH 7.5, before final dialysis into buffer B consisting of 50 mm sodium phosphate, pH 7.5, 10 mm magnesium chloride, and 50 mm potassium chloride. The GroEL monomer concentration was determined by absorbance measurements using ϵ280 = 12,200 liter mol–1 cm–1 (30Fisher M.T. Biochemistry. 1992; 31: 3955-3963Crossref PubMed Scopus (101) Google Scholar). Recombinant Shewanella methionine sulfoxide reductase B/A (MsrB/A) was a kind gift of Dr. T. C. Squier (Pacific Northwest National Laboratory, Richland, WA) (31Buchanan M.V. Larimer F.W. Wiley H.S. Kennel S.J. Squier T.C. Ramsey J.M. Rodland K.D. Hurst G.B. Smith R.D. Xu Y. Dixon D. Doktycz M.J. Colson S. Gesteland R. Giometti C. Young M. Giddings M. OMICS. 2002; 6: 287-303Crossref PubMed Scopus (6) Google Scholar). This protein contains both reductase domains necessary to reduce both methionine sulfoxide diastereomers, Met-(R)-SO and Met-(S)-SO. Sodium hypochlorite (NaOCl) and methane sulfonic acid was purchased from Aldrich and H2O2 from Fisher Scientific. Peroxynitrite was synthesized from azide according to the method of Pryor et al. (32Pryor W.A. Cueto R. Jin X. Koppenol W.H. Ngu-Schwemlein M. Squadrito G.L. Uppu P.L. Uppu R.M. Free Radic. Biol. Med. 1995; 18: 75-83Crossref PubMed Scopus (233) Google Scholar). Thioglo-1 was purchased from Covalent Associates (Woburn, MA). Sequencing grade modified trypsin was from Promega (Madison, WI). ortho-Phthalaldehyde was from Pierce. dl-Methionine, diethylamine/nitric oxide (DEA/·NO) complex sodium salt, catalase, N-acetylcysteine, sodium iodoacetate, dithiothreitol (DTT), porcine mitochondrial malate dehydrogenase (porcine mitochondrial MDH), ketomalonic acid, and β-NADH (reduced) were from Sigma. All other chemicals were from Fisher Scientific. Stock solutions of H2O2, –OCl (in 0.1% NaOH), and ONOO– were UV spectrophotometrically calibrated using ϵ240 = 39.4 liter mol–1 cm–1 for H2O2 (33Nelson D.P. Kiesow L.A. Anal. Biochem. 1972; 49: 474-478Crossref PubMed Scopus (820) Google Scholar), ϵ290 = 350 liter mol1 cm–1 (pKa at 25 °C is 7.537 ± 0.05) for –OCl (34Morris J.C. J. Phys. Chem. 1966; 70: 3798-3805Crossref Scopus (803) Google Scholar) and ϵ302 = 1,670 liter mol–1 cm–1 for ONOO– (35Hughes M.N. Nicklin H.G. J. Chem. Soc. (A). 1968; : 450-456Crossref Google Scholar). Nitric oxide was generated in situ by controlled release from the DEA/·NO complex at a ratio of 1.5 mol of ·NO/mol of DEA/·NO (36Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hoffman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (700) Google Scholar). Reactions were carried out in buffer B containing 1 mg/ml GroEL (17.5 μm monomer) and various concentrations of oxidant. All reactions were carried out at room temperature for 30 min except for the incubation with H2O2, which was run for 3 h, after which catalase (final concentration: ∼5nm) was used to quench residual H2O2. For HOCl and ONOO–, a final concentration of 5 mm Met was used to terminate the reactions. The activity of GroEL was determined through refolding of denatured MDH into the active form, according to the method by Tieman et al. (37Tieman B.C. Johnston M.F. Fisher M.T. J. Biol. Chem. 2001; 276: 44541-44550Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Briefly, MDH was denatured in buffer A containing 6 m guanidine hydrochloride and 8 mm DTT, for 1 h at room temperature. Refolding was initiated by rapid 100-fold dilution with buffer B (+components) resulting in the following concentrations: 1 μm (oligomer) native or oxidant-treated GroEL, 0.5 μm MDH, 2 μm GroES, and 5 mm ATP. The refolding reaction was carried out for 1 h at 37 °C before the enzymatic activity of MDH was determined using 1 mm ketomalonic acid and 0.2 mm NADH in buffer B (to 0.07 μm MDH). The rate of oxidation of NADH at 340 nm was followed for 3 min using a U3210 Hitachi (Tokyo, Japan) spectrophotometer. Native or oxidant treated GroEL was denatured with 6 m guanidine HCl in 18.75 mm sodium phosphate buffer, pH 7.5. Stock solutions of Thioglo-1 were prepared in dimethyl formamide and added to denatured GroEL samples to give a final molar ratio of 5:1 dye to free thiol (Cys). Calibration curves in the same concentration range were generated with N-acetyl-l-cysteine as a standard. Fluorescence yields were determined after 30-min incubation at room temperature using a Bio-Tek (Winooski, VT) FL600 Microplate Reader. Excitation and emission wavelengths were 360 and 530 nm, respectively. Hydrolysis was performed according to the method of Simpson et al. (38Simpson R.J. Neuberger M.R. Liu T.-Y. J. Biol. Chem. 1976; 251: 1936-1940Abstract Full Text PDF PubMed Google Scholar). Briefly, GroEL samples were hydrolyzed in vacuo with 4 n methanesulfonic acid at 115 °C for 22 h and neutralized with 3.5 n sodium hydroxide. Methionine sulfoxide was separated from other amino acids by HPLC after precolumn o-phthalaldehyde derivatization as described previously (39Sharov V.S. Ferrington D.A. Squier T.J. Schöneich C. FEBS Lett. 1999; 455: 247-250Crossref PubMed Scopus (165) Google Scholar). The o-phthalaldehyde derivatization reagent was prepared by mixing equal volumes of stock (25 mg of o-phthalaldehyde, 0.625 ml of methanol, 25 μl of 2-mercaptoethanol, 11.2 ml of 0.4 m K2HPO4, pH 9.5) with 1% Brij solution. The sample (10 μl) was mixed with 40 μl of the derivatization reagent for 1 min before adding 80 μlof 0.4 m KH2PO4. After another min, 100 μl of the sample was injected onto a Hypersil ODS column (4.6 × 250 mm; ThermoHypersil-Keystone, Bellefonte, PA). Methionine sulfoxide and Met were separated from the other amino acids at 35 °C at a flow rate of 1 ml/min using a linear gradient of 33–100% solvent B within 15 min, where solvents A and B were 95:5 (v/v) 25 mm sodium acetate (pH 5.85):tetrahydrofuran and 95:5 (v/v) methanol:tetrahydrofuran, respectively. Detection was by fluorescence (Shimadzu RF-10Asl; Shimadzu, Columbia, MD) with excitation at 330 nm and emission at 450 nm. The same separation conditions were also sufficient to resolve the o-phtalaldehyde-derivatized 3-chlorotyrosine from the other amino acids. Oxidant-treated GroEL samples were incubated with 1 μm MsrB/A and 15 mm DTT for 2 h at 37 °C. The final GroEL concentration was 15.8 μm in buffer B. Prior to tryptic digestion, native and oxidant-treated GroEL (starting concentration: 1 mg/ml) were reduced with 1 mm DTT in 6 m guanidine HCl for 30 min at 37 °C and alkylated with 3 mm sodium iodoacetate for 30 min at 37 °C. Subsequently, dialysis into 50 mm sodium phosphate buffer, pH 8.0, was carried out using Pierce Mini Dialysis Units, with the final buffer exchange containing 10% acetonitrile. Trypsin was initially added at a 1:20 molar ratio to GroEL. After 1 h at 37 °C, another aliquot was added to adjust the final trypsin to GroEL molar ratio to 1:10 (total trypsin to GroEL). At this stage, samples contained ∼0.4 mg/ml GroEL. After 18 h, the digestion was stopped by cooling to –20 °C. On-line HPLC-MS/MS Analysis—For on-line HPLC-MS/MS analysis we used a ThermoElectron LCQ-Duo mass spectrometer (ThermoElectron Corp., San Jose, CA) coupled to a gradient HPLC consisting of two Micro-Tech Ultra Plus II gradient pumps. Samples (10 μlof ∼0.4 mg/ml peptides) were injected onto a Vydac 218MS5.305 C18 column (300-Å pore diameter, 50 × 0.32 mm) equilibrated with 90% mobile phase A (99.9% ultrapure water/0.1% formic acid) and 10% mobile phase B (99.9% acetonitrile/0.1% formic acid). Gradient separation was achieved by a linear increase of the mobile phase ratio to 100% solvent B within 60 min and holding at this ratio for 5 min before returning to starting conditions. The following instrumental conditions were used for mass spectrometric analysis: number of microscans = 3, length of microscans = 200 ms, capillary temperature = 200 °C, spray voltage = 4.5 kV, capillary voltage = 14 V, tube lens offset =–17 V. Data acquisition was performed in the data-dependent fashion, i.e. a MS scan followed by MS/MS measurement with the normalized collision energy for MS/MS set at 35% and the isolation width of 2.0 m/z. A minimal signal for MS/MS acquisition was set to 2 × 105. Additionally, the dynamic exclusion option was enabled and set with the following parameters: repeat count = 3, repeat duration = 5 min, exclusion list size = 25, exclusion duration = 5, and exclusion mass width = 3. The sequence of native protein was matched using the TurboSEQUEST search option in the Bioworks Browser 3.1 software (ThermoElectron Corp.). Oxidatively modified sequences were matched manually by searching the data using the Xcalibur™ software package (ThermoElectron Corp.). The criteria for positive identification of a tryptic fragment were 1) matching of the observed m/z to the theoretical m/z and 2) matching of the collision-induced dissociation (CID) pattern to at least 3 consecutive fragment ions. Furthermore, confirmation of the location of modification in a peptide required the absence of the native residue. Off-line HPLC and MS/MS Analysis—For off-line mass spectrometric analysis, 50–100-μg aliquots of a tryptic digest were separated using a 5-μm 250 × 4.6 mm-Hypersil ODS column (ThermoHypersil-Keystone). The mobile phases used were 3% acetonitrile/97% water containing 0.1% trifluoroacetic acid (solvent A) and 60% acetonitrile/40% water containing 0.1% trifluoroaceric acid (solvent B). A gradient of 0–100% solvent B over 120 min was generated using two Shimadzu LC-10AS pumps. The flow rate was 1 ml/min with detection at 215 nm using a Shimadzu SPD 10AV UV/VIS detector. Peaks were collected, lyophilized, and stored at –20 °C. Dry samples were solubilized in 90% methanol, 0.5% formic acid and introduced into an electrospray ionization source from a 20 μl injector loop at 10 μl/min. Spectra were acquired on a Q-Tof-2™ hybrid mass spectrometer (Micromass Ltd., Manchester, UK). The instrument was operated for maximum resolution with all lenses optimized on the [M+2H]2+ ion from the cyclic peptide Gramicidin S. The cone voltage was 35 eV, argon was admitted to the collision cell at a pressure that attenuates the beam to about 20%, and the cell was operated at 8 eV (maximum transmission). Spectra were acquired at 11,364-Hz pusher frequency covering the mass range 100 to 3,000 atomic mass units and accumulating data for 5 s per cycle. Time-to-mass calibration was made with CsI cluster ions acquired under the same conditions. CID spectra were acquired by setting the MS1 quadrupole to transmit a precursor mass window of + 1.5 atomic mass units centered on the most abundant isotopomer. Argon was the collision gas admitted at a density that attenuates the beam to 20%; this corresponds to 16 p.s.i. on the supply regulator or 5.3 × 10–5 mbar on a penning gauge near the collision cell. The collision energy was varied from 20 to 35 eV to obtain a distribution of fragments from low to high mass. Spectra were acquired for 2–5 min in 5-s cycles. Spectra were acquired at 16,129-Hz pusher frequency covering the mass range 50–2,000 atomic mass units and accumulating data for 5 s per cycle. Chromatographic analysis of native and oxidant-treated GroEL was performed on a BIOSEP-SEC-S4000 300 × 7.8-mm column (Phenomenex, Torrence, CA) connected to a Shimadzu LC-10AS pump. The run buffer was 50 mm sodium phosphate, pH 7.5, 10 mm magnesium chloride, 50 mm potassium phosphate (buffer B), at 1 ml/min flow rate. Detection was by a Shimadzu SPD 10AV UV/VIS detector at 280 nm. The injection volume was 50 μl of 1 mg/ml GroEL sample. Samples were run on 4–15% Phast Gels (non-reducing conditions) using a Pharmacia (Peapack, NJ) LKB Phast System. Protein was monitored by Coomassie Blue staining. The plots displayed in Fig. 1A reflect the activity of GroEL at refolding denatured MDH following treatment with various concentrations of oxidant. MDH is a class III type substrate (2Netzer W.J. Hartl F.U. Trends Biochem. Sci. 1998; 23: 68-73Abstract Full Text PDF PubMed Scopus (196) Google Scholar), which requires the complete chaperonin system (GroEL and GroES) to refold. Any oxidation-dependent defects in the chaperonin system are more likely to show up with such a stringent substrate because of the multiple reactions that are necessary for a fully functioning chaperonin reaction (ATPase, GroES binding, GroES release, GroEL conformational changes, complex formation, polypeptide release). No significant inactivation of GroEL was observed when treated with 1 mm DEA/·NO for 30 min. Even treatment with 10 mm H2O2 for 3 h only caused 40% inactivation (data point not shown but was used to extrapolate data for 1 mm H2O2 in Fig. 1A). On the other hand, more than half of the activity of GroEL was lost when treated with either 0.5 mm ONOO– or 0.25 mm HOCl and ≥80% inactivation occurred with 1 mm ONOO– and 0.5 mm HOCl, respectively. The data demonstrate an efficient inactivation of GroEL by HOCl and ONOO– but negligible effects of ·NO and H2O2. We examined the potential reversibility of inactivation representatively for the exposure to HOCl (Fig. 1B). The inactivation of GroEL treated with 0.1–1.0 mm HOCl could not be reversed by reaction with 15 mm DTT alone (data not shown). In contrast, especially for GroEL treated with 0.1–0.25 mm HOCl, some of the lost activity could be recovered through exposure to 1 μm MsrB/A in the presence of 15 mm DTT. DTT is a suitable substitute for the physiological electron donor of methionine sulfoxide reductase, thioredoxin (40Brot N. Weissbach H. Biofactors. 1991; 3: 91-96PubMed Google Scholar). The incubation with MsrB/A recovered ∼70, 85, and 60% of the activity lost after treatment with 0.1, 0.175, and 0.25 mm HOCl, respectively. For HOCl concentrations ≥ 0.5 mm, the combination of MsrB/A with 15 mm DTT did not restore the activity. The activity of MsrB/A was independently confirmed through amino acid analysis of methionine sulfoxide after methanesulfonic acid hydrolysis of native and HOCl-treated GroEL. The exposure of GroEL samples to increasing concentrations of HOCl between 0.1 and 0.5 mm resulted in increasing yields of methionine sulfoxide which were restored to base-line levels through reaction with 1 μm MsrB/A and 15 mm DTT (data not shown). Fig. 1C displays the loss of free thiol residues in GroEL as a function of oxidant treatment. Clearly, the exposure to ·NO or H2O2 did not result in a significant loss of free thiols. In contrast, HOCl and ONOO– efficiently reacted with Cys. However, our results with MsrB/A suggest that by no means Cys oxidation alone can be responsible for protein inactivation. Analysis by On-line HPLC-MS/MS—Tryptic digests of native and oxidized GroEL (0.1, 0.25, and 1.0 mm HOCl; 0.5 mm and 1.0 mm ONOO–) were analyzed for oxidative modifications. For native GroEL we obtained a sequence coverage >85% matched to the primary sequence of GroEL (NCBI accession number NP_313151), as shown in Fig. 2. Table I summarizes all the detected modifications for the respective oxidizing conditions. We note that the employed mass spectrometric conditions did not allow for the quantitation of the protein modifications.Table ISummary of modified peptides detected by MS and MS/MS analysisFragment + modificationSequenceax indicates peptide identified by at least 3 consecutive fragment ions (MS/MS). ? indicates matching m/z but lack of unambiguous MS/MS. (O) indicates an oxygen addition at this position, characterized by an increase of the m/z of the native peptide by +16 Da. (O3) indicates the addition of three oxygens, characterized by an increase of the m/z of the native peptide by +48 Da. (NO2) indicates tyrosine nitration, characterized by an increase of the m/z of the native peptide by +45 Da. (Cys(Cm)) indicates carboxymethylcysteine, characterized by an increase of the m/z of the native peptide by +58 Da.mm HOCImm ONOO1.00.250.100.01.00.50.0T12 13+32EIELEDKFENM69(O)GAQM73(O)VKxxxxxT16+32AVAAGM111(O)NPM114(O)DLKxxxT16 17+32AVAAGM111(O)NPM114(O)DLKRxxx?T20+58ALSVPC138(Cys(Cm)SDSKxxxxxxxT20+48ALSVPC138(O3)SDSKx?xT22+16LIAEAM166(O)DKxxx?xT24+16EGVITVEDGTGLQDELDVVEGM193(O)QFDRxxxxx?T25+45GY199LSPY203(NO2)FINKPETGAVELESPFILLADKxxT28+16EM233(O)LPVLEAVAKxxx?xxxT35+32AM288LQDIATLTGGTVISEEIGM307(O)ELEKxxx??T34 35+32KAM288LQDIATLTGGTVISEEIGM307(O)ELEKxxxxx?T57+58QIVLNC458(Cys(Cm))GEEPSVVANTVxxxxxxxT57+48QIVLNC458(O3)GEEPSVVANTVxxxT58+16GGDGNY476GY478 NAATEEY485GNM488IDM491(O)GILDPTKxxxxxT58+32GGDGNY476GY478NAATEEY485 GNM488(O)IDM491(O)GILDPTKxxxxxT58+45GGDGNY476GY478NAATEEY485 GNM488IDM491GILDPTKxxT60+58SALQY506AASVAGLM514 ITTEC519(Cys(Cm))M520VTDLLPKxxxxxxxT60+" @default.
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• W2022025232 title "Potential Role of Methionine Sulfoxide in the Inactivation of the Chaperone GroEL by Hypochlorous Acid (HOCl) and Peroxynitrite (ONOO–)" @default.
• W2022025232 cites W1007804022 @default.
• W2022025232 cites W1523829738 @default.
• W2022025232 cites W1574128379 @default.
• W2022025232 cites W1591074156 @default.
• W2022025232 cites W1605950929 @default.
• W2022025232 cites W1835395104 @default.
• W2022025232 cites W1955167637 @default.
• W2022025232 cites W1964399631 @default.
• W2022025232 cites W1968072781 @default.
• W2022025232 cites W1968411230 @default.
• W2022025232 cites W1978515597 @default.
• W2022025232 cites W1981191309 @default.
• W2022025232 cites W1981992662 @default.
• W2022025232 cites W1988259156 @default.
• W2022025232 cites W1988587584 @default.
• W2022025232 cites W1991654844 @default.
• W2022025232 cites W1997678546 @default.
• W2022025232 cites W1998879311 @default.
• W2022025232 cites W2000069699 @default.
• W2022025232 cites W2006834407 @default.
• W2022025232 cites W2006903305 @default.
• W2022025232 cites W2011667964 @default.
• W2022025232 cites W2012634726 @default.
• W2022025232 cites W2013204959 @default.
• W2022025232 cites W2019730574 @default.
• W2022025232 cites W2029314163 @default.
• W2022025232 cites W2031469201 @default.
• W2022025232 cites W2035961359 @default.
• W2022025232 cites W2039042657 @default.
• W2022025232 cites W2040447429 @default.
• W2022025232 cites W2046500133 @default.
• W2022025232 cites W2067766350 @default.
• W2022025232 cites W2074272236 @default.
• W2022025232 cites W2074314874 @default.
• W2022025232 cites W2075034126 @default.
• W2022025232 cites W2077492725 @default.
• W2022025232 cites W2088938488 @default.
• W2022025232 cites W2088971532 @default.
• W2022025232 cites W2090316954 @default.
• W2022025232 cites W2096966529 @default.
• W2022025232 cites W2098501605 @default.
• W2022025232 cites W2099757932 @default.
• W2022025232 cites W2101819482 @default.
• W2022025232 cites W2104611484 @default.
• W2022025232 cites W2107993295 @default.
• W2022025232 cites W2117782167 @default.
• W2022025232 cites W2119928987 @default.
• W2022025232 cites W2120620942 @default.
• W2022025232 cites W2121456628 @default.
• W2022025232 cites W2132296203 @default.
• W2022025232 cites W2133536720 @default.
• W2022025232 cites W2134502101 @default.
• W2022025232 cites W2145410445 @default.
• W2022025232 cites W2158604239 @default.
• W2022025232 cites W2314530439 @default.
• W2022025232 cites W32138488 @default.
• W2022025232 cites W4229630303 @default.
• W2022025232 cites W4253408758 @default.
• W2022025232 cites W90004733 @default.
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